ABSTRACT Persistent immune activation is the defining feature of HIV-1 infection in vivo and a driver of progression to end- stage AIDS. Systemic immune activation in people living with HIV has been hypothesized to account for higher incidence of chronic inflammatory diseases, including HIV-associated neurocognitive disorders (HAND). Importantly, numerous studies have demonstrated that populations living with opioid-use disorder (OUD) have a higher occurrence of comorbid HIV infection, suggesting both potential socio-economic and neurocognitive influence. This comorbidity of OUD and HIV has also been shown to increase neuroinflammation and exacerbate the progression of HIV infection and HAND. While some studies have investigated the interactive effects of OUD and HAND in few cell types in isolation, the molecular mechanisms linking the two conditions is still largely unknown, especially in primary human microglia, neurons and astrocytes. Hence, we believe that this proposal fulfills a critical unmet need by establishing primary human neuronal cell cultures and an unbiased, systematic single cell transcriptomic survey of these cultures in the context of co-exposure of opiates and HIV-1 infection. In this proposal, we will establish 3D spheroid cultures consisting of induced pluripotent stem cell (iPSC)-derived human microglia, astrocytes and neurons, derived from 10 independent donor lines established at BUMC, that will recapitulate spatial complexity of cell-to-cell interactions in the brain, and interrogate the effect of HIV infection and morphine exposure on persistent innate immune activation. We will utilize cutting-edge single cell transcriptomics analysis to identify the microglia-intrinsic pathway that promotes induction of pro-inflammatory responses upon co-exposure to HIV and opiates and the neurodegenerative pathways contributing to neuronal cell injury. Finally, information from these transcriptomic studies will be leveraged to inform CRISPR/Cas-based knock-out strategies to selectively delete key pathways in iPSC-microglia to alleviate neuroinflammation. We believe that results from these studies will inform future therapeutic development to prevent HIV and opiate- induced neuroinflammation and prevent development of HAND in HIV+ substance use population.